This work involves general characterization of enzymes involved in respiration. The subject organism is the thermophilic bacterium Thermus thermophilus. During past funding periods we have established a working foundation in the biology, biochemistry, and molecular biology of this organism. With this knowledge it has been possible to carry out a wide range of biochemical, biophysical, and physical characterizations of its respiratory proteins. Of great importance is the recent conclusion from gene sequencing work that the respiratory proteins from this organism are functional and structural homologs of eukaryotic counterparts. Indeed, analogs of the bacterial enzymes appear to form the core of eukaryotic enzymes. For this reason, the results of our structure-function studies are directly applicable to all other such enzymes, notably those of the mammalian mitochondrion. During the past funding period; we purified two new proteins, cytochromes ba3 and b562; cloned and sequenced the genes encoding cytochromes c552 and caa3; carried out detailed physical studies of the CuA and a3/CuB sites in cytochromes ba3 and caa3; examined the Rieske iron-sulfur center using a combination of stable isotope labeling and ENDOR spectroscopy; and established the rudiments of a molecular biology for Thermus in our laboratory. The current proposal suggests a continued wide-ranging experimental approach with five major scientific goals: [1] Establish the relationship of cytochrome ba3 to the greater family of heme/copper oxidases. [2] Count all suspected histidine and cysteine ligands to the canonical metals of cytochrome oxidase by using a combination of isotope labeling and resonance spectroscopies. [3] Obtain active site structural details of the respiratory-type Rieske protein. [4] Determine the electronic structure of the oxidized a3/CuB metal pair. And, [5] examine the effect of transmembrane potential on the vibrational properties of cytochrome c oxidases. We also suggest two major technical goals to expand the operational base of the project: [1] Attempt to obtain single crystals of several respiratory proteins, and [2] establish a plasmid vector for the expression of Thermus genes in their natural environment. Over the past several years, there has been increasing recognition that inherited defects in the mitochondrial respiratory chain are important in human diseases. The significance of this work to human health lies in a deeper understanding of the enzymes that provide energy to all cells.